The objective of this proposal is to identify cellular proteases and viral determinants of proteolysis that regulate reovirus cell entry and to understand how these factors impact pathogenesis. In Aim 1, we will identify proteases that mediate productive reovirus uncoating. We will focus on those that are likely to be physiologically relevant, including serine proteases present in the respiratory tract, and cathepsin S, which is expressed by macrophages. In Aim 2, we will use genetic and biochemical approaches to identify sequences within capsid protein sigma 3 that regulate cell entry. We will select and characterize viral mutants with expanded protease-sensitivity, and we will use particles recoated with recombinant sigma 3 to identify determinants of protease susceptibility and cell entry. In Aim 3, we will use mouse infection models to investigate the role that specific intracellular and extracellular proteases play in regulating reovirus tropism, spread and disease. We will compare virions and uncoated particles to determine whether the requirementfor sigma 3 proteolysis represents a barrier to reovirus infection in the respiratory tract. Using knockout mice, we will ask whether the cathepsins L and S, which are determinants of infection in cell culture, also play critical roles in vivo.We will also investigate the importance of the serine proteases cathepsin G and elastase for infection at extraintestinal sites. Our studies of the molecular determinants of reovirus cell entry will provide fundamental information concerning the biology of non-enveloped viruses. They will positively impact human health by facilitating the safe and effective use of reovirusesas oncolytic agents to treat human tumors.